Additional file 1: Figure S1. of Acute chagas outbreaks: molecular and biological features of Trypanosoma cruzi isolates, and clinical aspects of acute cases in Santander, Colombia

Genotyping of T. cruzi isolates. Mini-exon intergenic spacer, 24Sα rRNA, 18S rRNA, glucose-6 phosphate isomerase (GPI), heat shock protein 60 (HSP60), and mitochondrial gene Cytochrome Oxidase subunit II (COII) genes were characterized. (TIF 386 kb

Characterization of a novel antibiofilm effect of nitric oxide-releasing aspirin (NCX-4040) on <i>Candida albicans</i> isolates from denture stomatitis patients

<div><p><i>Candida albicans</i> biofilms play a key role in denture stomatitis, one of the most common oral pathologies in elderly people. Because biofilms are highly resistant to antifungals, new pharmacological strategies are needed. Aspirin and nitric oxide-donor molecules have both shown antibiofilm effects on <i>C</i>. <i>albicans</i>, making them promising candidates for treatment. In this study, we evaluated the antifungal/antibiofilm effect of a nitric-oxide releasing aspirin (NO-ASA) on <i>C</i>. <i>albicans</i> isolates from denture stomatitis patients <i>in vitro</i>. Disk diffusion assays showed that while NO-ASA had no antifungal effect, the drug potentiated fluconazole inhibition zone diameters, increasing the effect of fluconazole by 20–30% (p<0.05). The effect of NO-ASA on the morphogenesis of <i>C</i>. <i>albicans</i> was evaluated using light microscopy after inducing hyphae formation. For all clinical strains assayed, 125 μM NO-ASA significantly decreased the number of filamentous cells present (p<0.01). Adhesion to abiotic surfaces, a critical event for biofilm formation, was evaluated in 96-well polystyrene plates using crystal violet assay; 125 μM NO-ASA significantly inhibited adhesion. Biofilms were observed with scanning electron microscopy (SEM) and quantified using XTT reduction assay. NO-ASA decreased biofilm formation (IC₅₀ ranging from 300 μM to 700 μM), consistent with SEM findings of altered biofilm microarchitecture. PGE₂ and carboxy-PTIO (an NO scavenger) both blocked the antibiofilm effects of NO-ASA, suggesting that the efficacy of NO-ASA may be associated with both inhibition of PGE₂ synthesis and release of NO. NO-ASA is a promising novel antibiofilm agent for treating fluconazole-resistant strains of <i>C</i>. <i>albicans</i>.</p></div

Nitric Oxide scavenger prevents NO-ASA antibiofilm effect.

<p>Biofilms were pretreated with 200 μM carboxy-PTIO for 20 min at 37°C. Then NO-ASA 250 μM or 1 mM was added and biofilms were incubated for 24h at 37°C. Bars represent mean ± SD of biofilm formation quantified trough XTT reduction assay. * p<0.05; ** p<0.01; *** p>0.001; ****p>0.0001 as compared with control (DMSO). †p<0.05; ††p<0.01; ††† p>0.001; ††††p>0.0001 as compared between groups as indicated (one way ANOVA).</p

PGE₂ prevents the antibiofilm effect of NO-ASA.

<p>24-h biofilms were incubated with 250 μM or 1 mM NO-ASA for an additional 24 h in the presence or absence of exogenously-added PGE₂. Bars represent the mean ± SD biofilm formation quantified through XTT reduction assay. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 as compared to NO-ASA-treated biofilms in the absence of PGE₂.</p

Biofilm microarchitecture is affected by NO-ASA treatment.

<p>Representative SEM analysis of 17p strain biofilms on coverslips in the presence or absence of 1 mM NO-ASA. Control biofilms were composed of a dense layer of filamentous cells <b>(A, B)</b>. Treatment with NO-ASA reduced the density of the biofilm layers and decreased the presence of filamentous cells <b>(C, D)</b>.</p

NO-ASA inhibits <i>C</i>. <i>albicans</i> planktonic morphogenesis.

<p><i>Upper Panel</i>: light microscopy photographs showing representative filamentous cells (hyphae or pseudohyphae) for the 17p strain after 3 h or 12 h of incubation with DMSO <b>(A, E)</b>; 800 μM fluconazole <b>(B, F)</b> or 500 μM aspirin <b>(C, G).</b> Cells treated with 500 μM NO-ASA <b>(D, H)</b> showed mainly budding yeast cells and scarce filamentous cells (arrow in H). <i>Lower panel</i>: Bars represent mean ± SD of percentage of filamentous cells as compared to untreated controls for each strain assayed after 3 hours of incubation (untreated control bars were omitted for clarity). ****p<0.0001 as compared to fluconazole alone (white bars); †p<0.05; †††p<0.0001 for comparisons between treatments as indicated; ns = no significant difference (two-way ANOVA).</p

NO-ASA increases the antifungal effect of fluconazole in resistant strains.

<p>Bars represent mean ± SD of inhibition zone diameters. Discs contained 25 μg fluconazole, alone or in combination with 25 μg nitric oxide-releasing aspirin or 25 μg ASA. Controls containing only vehicle (DMSO), aspirin (ASA), or NCX-4040 NO-ASA showed no inhibition zone diameters (not shown). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 as compared to fluconazole alone; †p<0.05; ††p<0.01 for comparisons between treatments as indicated; nd = inhibition zone not detected (two-way ANOVA).</p

NO-ASA inhibits the adhesion of <i>C</i>. <i>albicans</i> to abiotic surfaces.

<p>Bars represent mean ± SD of percentage of cells adhered to the bottom of wells as compared controls for each strain assayed. Due fluconazole 800 μM did not affect adhesion as compared to untreated strains it was used as control for the analysis. **p<0.01; ****p<0.0001 as compared to fluconazole alone (white bars); ††††p<0.0001 for comparisons between treatments as indicated; ns = no significant difference (two-way ANOVA).</p

IC₅₀ values of NO-ASA for <i>C</i>. <i>albicans</i> biofilms from denture stomatitis isolates.

<p>IC₅₀ values of NO-ASA for <i>C</i>. <i>albicans</i> biofilms from denture stomatitis isolates.</p

Effect of 15-epi-LXA₄ on the outcome of experimental Chagas' disease in ASA-treated mice.

<p><b>A.</b> Survival of mice infected with <i>T. cruzi</i> (Dm28c strain) treated with ASA (75 or 100 mg/Kg) with or withouh 15-epi-LXA₄ (25 mg/Kg). Survival was recorded daily from day 1 p.i. Graph representative from two independent experiments (n = 6 each). <b>B.</b> Real-time PCR analysis of heart tissue from infected and treated mice. Hearts were extracted at the dead day, or on day 20 p.i. for survival mice. Each point represents one mouse. Discontinuous line indicates the mean from at least 5 mice. The results are expressed as the normalized DNA ratio, relative to control. <b>C.</b> Parasitemia of <i>T. cruzi-</i>infected and treated mice. To facilitate visual assessment, separate doses of ASA are showed in different panels. For each graph, black square represent control groups, black circles represents mice treated only with ASA and white circles represent the group treated with ASA and 15-epi-LXA₄. All measurements were made with n = 6 mice per group. **: p<0.01 and ***: p<0.001, as compared to control and ¶: p<0.01 compared with corresponding ASA-treated group, calculated by two-way ANOVA. <b>D.</b> Histological examination of hearts from <i>T. cruzi</i> infected and treated mice. Hearts were extracted at the dead day, or on day 20 p.i. for survival mice. Hearts were fixed in 10% formaldehyde and embedded in paraffin. Slides were stained with hematoxylin-eosin. Black arrowheads indicate amastigote nests Images represent at least three mice in each group. <b>E.</b> Infiltrate quantification from histopathological analysis. Cells nuclei of infiltrate were quantified by using the ImageJ software. The graph shows the mean ± SD of at least five mice with three non-consecutive slides each. *: p<0.05 compared to control, and **:p<0.01 compared with ASA 75-treated group, calculated by two-way ANOVA and Tukey post-test.</p